JPH05507924A - immunoreactive compounds - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] immunoreactive compounds The present invention relates to immunoreactive compounds and pharmaceutical preparations containing the same.

Such immunoreactive compounds are particularly suitable for use in immunotherapy and diagnostic methods. I can do it.

Immunotherapy is one of the promising possibilities for combating numerous diseases.

The principles of immunotherapy itself are old. This involves placing the active substance in close proximity to the target. There is a targeting component to be transported.

Therefore, we define this as a group of cells that share a site where the targeting moiety is available. It can be used to kill or optionally stimulate the spores. ligand -Receptor interaction or antibody-antigen interaction is the relationship between the target and the target. Of course, others will occur to those skilled in the art. It will probably be a hit.

Perhaps more sophisticated methods of immunotherapy are the so-called pre-targeted techniques. child These include, but are not limited to, precursor drug activation, which conjugate to a targeting component, making this targeting component less toxic than its loaded drug. be administered before or with the precursor drug, and the enzyme targets the precursor drug. It is converted into a loaded drug at the target site.

For anti-abscess therapy and tumor localization and general applications for cancer therapy, They are thought to be antibodies conjugated to labeled substances. anti-tumor therapy For example, such labeled products include adriamycin, valcarin, and california. Toxic compounds such as caremicin, mitomycin, ricin, or other suitable toxic compounds or isotopes or enzymes as described above. I can do it.

Antibodies are generally targeted to specific antigens of tumors. many cases The antibodies used for this purpose are monoclonal antibodies of mouse origin. It is a class of bodies. Mouse monoclonal antibodies are easily produced according to well-established methods. simply obtained and directed against virtually any antigen.

Nevertheless, these antibodies have a number of drawbacks. Mathematically removes tumor material. When administered to mice, they contain tumor material, including the normal antigens of these cells. produces antibodies against almost any antigen. In this way, it is specific to tumor cells only. It is difficult to obtain specific antibodies. Therefore, mouse antibodies Depending on the scope of the response, it may be directed against epitopes that are not tumor-specific.

Furthermore, the use of murine antibodies is hampered by their inherent immunogenicity in humans. Ru. We have developed a solution for the use of antibody fragments that mainly contain the antigen-binding region of mouse antibodies. This can overcome the secondary problem, but it does not solve the primary problem. There shouldn't be any.

A more ideal solution would be the use of human anti-tumor antibodies. Human anti-tumor antibodies , according to the method described in EP 0151030. Can be done. However, the problem is that this method mainly deals with IgM and/or IgA It is the production of immunoglobulins. Actually these are pentamers or are dimers, i.e. they are 5 linked together through S-8 bridges. Although composed of one or two monomers, each of these IgM/rgA monomers It consists of two heavy chains and two light chains, and has two antigen-binding sites. these Each of the monomers is roughly equivalent in size to an IgG molecule. therefore, A complete IgM molecule is approximately five times the size of an unprocessed IgG molecule. Most I gM has antigen-affinity properties that place it at the lowest end of the IgG affinity range. Hit.

The relatively large volume of IgM molecules makes it suitable for immunotherapy in vivo. They are not well suited for tumor imaging applications and tumor image projection, i.e. they are It takes a relatively long time to reach the purification value of unbound IgM, and the It takes at least five times longer than the other methods. How much is IgM? The same can be said for IgA, although not as much.

A straightforward solution is to fragment the IgMS or IgAZ polymer into monomers. It seems that there is. However, the antigen affinity of IgM/IgA monomers remains unknown. It has been reported that the affinity of IgG/IgA for treatment is dramatically lower than that of IgG/IgA. This is a difference of at least about 100 to 1.000 times.

This low affinity makes IgM/IgA monomers unsuitable for therapeutic and diagnostic applications. It has become. A similar decrease in affinity was observed for IgM obtained using enzymes. and fragments of IgA.

The present invention relates to restoration of antigen affinity of fragments of IgM. According to the invention, antigen binding The affinity of synthetic 1gM or IgA fragments is determined by linking them to at least one polypeptide. It can be restored by covalently bonding to peptides.

Such polypeptides conveniently contain human serum albumin or It can be a human protein, such as a (human) enzyme, or a poly-L- Synthetic polypeptides with low immunogenicity such as glutamic acid or poly-L-lysine It can be a peptide. Either IgM or IgA fragments are monomeric IgM or IgA monomer that can be obtained by reduction of the S-8 bond of or by the use of pepsin or papain, for example. Antigen-binding fragments obtained after enzymatic cleavage of IgM or IgA can. Digestion with pepsin is commonly denoted as F(a b’)2. produces an antibody fragment that further comprises two antibodies linked together by an S-8 bond. It forms the antigen-binding portion.

Reduction of these bonds produces two F(ab') fragments.

Ideally, both IgM or IgA monomer and F(ab') fragment can be attached to polypeptide(s) through their sulfur atoms.

However, IgM binding to polypeptides has antigen-binding properties. It also holds true with any other combination unless prevented. Regarding this, Glico If the zyl group is present in the constant region of the IgM or IgA fragment, It is also convenient to establish a bond.

The bond between the IgM or IgA fragment and the polypeptide is Either direct or indirect binding via a pacer Can be done.

Binding between IgM or IgA fragments and polypeptides is determined for both components. , bond, linker and/or spacer on one or both of the linking groups. Any group suitable for the desired reaction and subsequent reaction of those components may be utilized. can be established to form the desired immunoreactive compound. can.

Before or after binding to the IgM or IgA fragment, the polypeptide optionally with one or more therapeutically or diagnostically useful groups. can be converted into a file.

Suitable therapeutically useful groups include, for example, cytotoxic drugs, (optionally chelated) conversion of radioactive elements or precursor drugs into active drugs. These enzymes are used to

However, the polypeptide converts the precursor drug into a drug at the target site. If the enzyme itself is able to react, the size of its immunoreactive compounds Additional benefits exist because they play an important role in usability. proper diagnosis Radioactive groups are, for example, radioactive elements (optionally chelated). Ru. IgM or IgA fragments are conveniently human IgM or IgA fragments. Retrieved from gA. This IgM or IgA is tumor-specific or It is directed against antigens derived from tumor cells, which can also be expressed inside or on the surface of tumor cells. It can be found either on the outside or on the outside.

Example I A. Preparation of immunoreactive 1 gM monomer Human monoclonals against severe injury-associated epitopes present in colorectal cancer IgM antibodies 16-88 were converted to monomers by reduction with cysteine.

I gM (3-5 mg/m +) in PBS (6.7 mmol/l K/ Contained in Na phosphate buffer, p) (6,5; 0.13 mol/l NaCI) The cells were incubated for 3 hours at 37° C. in 10 mmol/I cysteine.

The buffer was saturated with nitrogen and the reaction vessel was sealed with gas.

After incubation, the reaction mixture was diluted with 1 mmol/l in PBS. Chromatography on Sephadex G25 equilibrated with cysteine I put it on. The monomers were precipitated with 50% saturated (NH)SO2 and further precipitated with pH 7, Cysteine was dissolved in a minimum volume of 1 mmol/l of cysteine contained in PBS of 5.5 mmol/l.

This monomer solution was mixed with 1 mmol/l of cysteine in PBS, pH 7.5. The resulting mixture was applied to a Fractogel TSKHW55 (S) column equilibrated with NaCl. This card The support volume of the ram is 45 times the volume applied to the column, and the elution rate is 0.06 Support volume/hour. The monomer usually has a K at the main A280 peak. Eluted at av=0.55-0.60.

They were precipitated with 50% saturated (NH)SO2.

The precipitate was dissolved in 0.1 mol/l sodium phosphate, O', 1 mol/l NaCl. , 5 mmol/l EDTA, 1 mmol/l cysteine, pH 7.5. After dissolution, the remaining ammonium sulfate was removed using the PBS buffer described above. Removed by gel filtration on Sephadex G25 equilibrated in EDTA. Ta.

Monomer of solid DTNB (dithionitrobenzoic acid, Ellman's reagent), desalted Add to the body-containing fraction to a final concentration of 20 mmol/l, and After gentle shaking, the reaction mixture was incubated for 3 hours at room temperature.

excess reagents and low molecular weight reaction products with 0.1 mol/l sodium phosphate; 0.1 mol/l NaCl; 5 mmol/l EDTA, pH 7.5 (solution A) In the medium, it was removed by gel filtration on Sephadex G25.

B. Reduction of ISA (-DTPA) H3A (-DTPA) was added to a concentration of 5-10 mg/m+ at 0.1 molar concentration. mol/l sodium phosphate; 0.1 mol/l NaCl; 5 mmol/l ED Dissolved in TA1 pH 7.5. For this solution, add DTT (dithiothlate) ) was added to a final concentration of 20 mmol/1 at room temperature. Incubation was performed for minutes. This reaction mixture is then mixed with excess reducing reagent. EDTA/PBS, as previously described, for the purpose of removing drugs and low molecular weight reaction products; Chromatography on Sephadex G25 equilibrated in pH 7.5. (Solution B).

C, IgM-)(SA(-DTPA) Immune Complex Preparation H3A(-DTPA) ) immediately after reduction of solution A (containing activated monomers) and solution B (reduced I (including SA) with a volume ratio of this monomer/ISA (-DTPA) of approximately 0.5. and mixed. Incubation was carried out overnight at room temperature.

After completion of the conjugation reaction, the unconjugated ISA (-DTPA) was added to 50% saturated (NH 4) Removed by 2S04 precipitation. Precipitated complexed and uncomplexed monomers Nono, ISA(-DTPA) remains in solution. This precipitate was 50% saturated. Wash several times with hydrated (NH) SO2 and add a minimum volume of 0.1 mol/l sodium phosphate. Thorium, 5 mmol/l, was dissolved in EDTAS pH 7.5. This solution, E D T A Equilibration in IJ acid buffer (buffer A) (without NaCl) Chromatography was performed on Sephadex G25. Then the protein The immunocomplexes were analyzed using anion exchange chromatography. For the purpose of separating uncomplexed monomers from (Fast Flow). After placing the sample, A280 is at the standard value level. The Q-Sepharose column was washed with buffer A until recovered.

Proteins retained on the column are converted to O-0.6 mol/1 (NaCl) by Na It was eluted by a stepwise method of increasing C1 concentration.

Although a group of non-complex malleable bodies passed through the column in the pass-through fraction, the immune complex The body is retained due to the acidic properties of ISA (-DTPA) and contained in buffer A. Eluted with o, ao, 4 mol/1 NaCl. on Sephadex G25 After desalting of the immune complexes, the final preparation was subjected to filtration through a 0.20 μm membrane. It was further sterilized and then stored in small aliquots until use.

Example 2 Preparation of IgM Fab'-ISA (-DTPA) complex IgM It was digested with pepsin according to the method of s■. Briefly, 091 mol/1 sodium 1 gM total at a concentration of 2-5 mg/ml in acetate buffer, pH 4.0. , incubate with pepsin (0.08-0.2 mg/ml) for 8 hours at 4°C. I started. This reaction mixture was combined with F(ab') formed from 1 gM total undigested ) For the purpose of purifying the 2 fractions and low molecular weight fragments, the fractions equilibrated in buffer A were Chromatography was performed on Kutogel HW55S. In this way, 40-60% recovery of mattographically pure F(a b’)2 isolated at a high rate.

F (a b') 2 is reduced with DTT, and the method described for the monomer was bound to ISA (-DTPA).

Purification of F(ab’)-ISA(-DTPA) was performed using Q - Performed by anion exchange chromatography and gel filtration on Sepharose did. The final preparation was sterilized by filtration and stored at 4°C.

IgM monomer-ISA (-DTPA) and IgM Fab'-ISA (-DTPA) - Immunoreactivity of immune complexes Immunoreactivity is determined by antigen binding assay. or competitive EIA.

In the antigen binding assay (dot-plot EIA), the Transfer a series of dilutions to the imopilone membrane in a Bio-Rad Transprot device. let Excess protein binding sites were blocked with 5% skim milk powder, and then the protein lot with peroxidase-labeled antigen contained in BPS buffer. Incubate. After a 2-hour incubation run at room temperature, the antigen-containing solution was discarded, the plot was washed three times with PBS-Tvreen, and the plot was further washed with enzyme polynucleotide. 2 mmol/l hydrogen peroxide and 0.6 mg/m as hydrogen acceptor. 1 of diaminobenzidine, 0.6 mg/ml of CoC12. put out A purple dot is visualized 5 minutes after the incubation run. The color intensity is measured by scanning on a Bio-Rad gel scanner.

In competitive EIA, a series of dilutions to be tested are peroxidase-labeled. microorganisms coated with a constant amount of 1 gM total and 0.1 μg/ml antigen solution. Incubate for 3 hours at room temperature in a titer plate. incubation After discarding the contents of each well, the plate was further washed with PBS-Tween. Wash 3 times with buffer. Enzyme activity is based on tetramethylbenzidine as a hydrogen acceptor. Detection is performed using a substrate solution containing This enzymatic reaction was stopped with 2 mol/I HS04, Read absorbance at 450 nm.

Both assays measure the immunoreactivity of IgM, IgM fragments, and immune complexes. Provides relative results regarding.

The purified monomer is 0.001-0.05 times more specific than the untreated total 1 gM. Although it has immunoreactivity (=IR per mass), F(a b')2 fragmentation The fragments are not immunoreactive in the assay applied. However, monomer The purified H8A (-DTPA) complexes were compared with those of the purified monomers. Then, total 1 gM and F(ab')-H8A ( -DTPA) exhibits immunoreactivity similar to that of the complexes.

In conclusion, for example through disulfide or thioether bridges (carrier ) Covalent attachment of proteins increases the immunoreactivity of IgM monomers or fragments. can be recovered considerably, to the greatest degree in the case of monomers .

(Enzyme-)conjugates can be prepared in two ways: A: 5PDP-based on local conjugation to activated enzyme B: This is due to the activation of the monomer with DTNB and the subsequent reaction with the enzyme. , has an unreacted -3)I group.

4.1. Method A Enzyme (0.1M N a 2 HP O4 / N a H2P O4, I) N 7, sho, 1/10 of 10mg/m+) contained in LM NaCl Add a volume of 40mM 5PDP (dissolved in absolute ethanol) and further Then, incubation was performed for 30 minutes at room temperature in the dark. This reaction mixture The sample was purified on a Sephadex G-25(M) column equilibrated with the same buffer. Unreacted 5PDP was removed by chromatography. 5PDP activated enzyme was added to the IgM-monomer in a ratio of 1:1 (w/w). Immediately before addition, the cells were incubated with Sephadex G-25 (M) equilibrated with this buffer. (It was subjected to romatography.)

The reaction mixture was incubated for 16 hours at room temperature in the dark. complex was recovered by addition of an equal volume of 100% saturated ammonium sulfate. Precipitate washed three times with 50% saturated ammonium sulfate, then dissolved in an appropriate buffer. I understand.

(Note: Although these final steps can be performed using HRP as the enzyme, , other purification methods may be required for other enzymes).

4.2. Method B Enzyme activation: First, activate the enzyme with 5PDP as described in Method A. Ta. This activated enzyme was treated with O, LM NaAc5 pH 4,5; Chromatography on Sephadex G-25 (M) equilibrated in NaCl - I called E. Add 1/20 volume of IM DTT to the enzyme-containing fraction. Incubation was performed for at least 30 minutes at room temperature in the dark.

Immediately prior to binding, this mixture was separated into a safe buffer for the preparation of the IgM-monomer-enzyme complex. Chromatography was carried out through Addex G-25 (M) process.

4.3. Monomerization (Human) IgM at 37°C in the solution shown below (saturated with N2). The monomerization was carried out by incubating with Aoki for 3 hours.

5mM NaHPO/NaHPO4, pH 6, 565mM NaCl 0.1g/I N a N 3 10mM cysteine 0.5 units papain/g TgM l-Log/h of IgM After this incubation, add an equal volume of 100% saturated ammonium sulfate solution. Added.

at 4°C (after standing for 2 hours and centrifuging to collect the precipitate), Dissolved in the following solution: 50mM Tris-HCl, pH 8. 140mM NaC] 1mM cysteine This solution was diluted in the same buffer to remove any remaining ammonium sulfate. Chromatography was carried out through a Sephadex G-25 (M) column. .

4.4. Purification of 1gM monomer The above solution was applied to a Fractogel HW-55(S) column (equilibrated in the same buffer). The mixture was subjected to chromatography through a

Fractions containing monomeric IgM (usually the second peak) were combined and added to 1 mM Addition of an equal amount of ammonium sulfate to 100% saturation in cysteine solution It was concentrated by This precipitate was collected and used for conjugation.

4.5゜Activation of monomeric IgM Monomeric IgM was passed through Sephadex G-25(M) using the solution shown below. and chromatography: 50mM Tris-HCl, pH 8. 140mM NaCl 1mM cysteine To this solution, add 15 mg of DTNB/ml solution in solid form. ), dissolved. Incubate the reaction mixture for 16 hours at room temperature in the dark. I did it. Sephade equilibrated in unreacted pH 7, 5, 0, 1M NaCl It was removed by chromatography through G-25(M).

The degree of activation of monomers with TNB can be determined in the following method. : 28G E 330 Concentration factor of TNH □ (II) (=B) TNB/monomer = B/A Complexing: Activated HRP and activated monomeric IgM were combined in a ratio of 1:1 ( They were added in each manner at a ratio of W/W). Incubate at room temperature in the dark. I went there for 16 hours. After this period, the complex was processed as described in Method A. Purified.

4.6 Immunoreactivity of the enzyme complex was detected with monoclonal antibodies 16.88 and 81AV78. coated with a crude antigen mixture of tumor cell lines expressing more recognized antigens. Determined by incubation of the complex in microtiter plates (Fig. 1). Monoclonal antibody 16.88 is directed against cytokeratins. While monoclonal antibody 81 specifically recognizes tumor-associated epitopes in MV78 reacts with tumor-associated antigens on the surface of cells.

However, an unrelated drug that has no reactivity against tumor cells or cell lines A complex of relevant antibodies, myeloma IgM, is produced against unpurified antigen preparations. Did not combine.

Myecromas are antibodies (antibodies) that recognize antigens that are not present in a mixture of unpurified antigens. IgM).

Binding assay Plates coated with unpurified antigen rig/ml-complex Binding assay Plate coated with CrAl7 ) direction complex summary to carrier molecules such as H3A, enzymes, or synthetic polypeptides of low immunogenicity. binding of one or more antigens of IgM or IgA covalently bound to A new class of immunoreactive compounds including sex fragments is provided. These fragments are placed on a carrier. Their relatively low antigen affinity can be overcome by covalently binding natural Ig The affinity level is restored to a level compared to that of M or IgA.

international search report international search report EP 9101223 SA 48769 international search report EP 9101223 SA 48769

Claims (11)

[Claims] 1. of IgM or IgA bound to at least one polypeptide Immunoreactive compounds, including antigen-binding fragments. 2. 2. An immunoreactive compound according to claim 1, wherein the fragment is a human IgM compound. or IgA. 3. Immunoreactive compound according to claims 1-2, comprising IgM or 1g The immunoreactive compound as described above, wherein A is directed against a tumor-associated antigen. thing. 4. An immunoreactive compound according to any one of claims 1 to 3, comprising: The immunoreactive compound as described above, wherein the peptide is a human protein. 5. An immunoreactive compound according to any one of claims 1 to 4, comprising: The peptide has the property of being human serum albumin or a fragment thereof, Immunoreactive compounds as described above. 6. An immunoreactive compound according to any one of claims 1 to 4, comprising: The immunoreactive compound described above, wherein the peptide has the property of being an enzyme. 7. 7. An immunoreactive compound according to any one of claims 1 to 6, comprising: The property that the peptide is additionally bound to one or more labeled substances The immunoreactive compound as described above. 8. An immunoreactive compound according to any one of claims 1 to 7, comprising the fragment is a monomer of IgM or IgA, Compound. 9. An immunoreactive compound according to any one of claims 1 to 8, comprising the fragment is an F(ab')2 fragment of IgM or IgA, Immunoreactive compounds. 10. A therapeutically active compound comprising an immunoreactive compound according to any of claims 1 to 9. a composition comprising at least one tumor ricin-like compound bound thereto; , the above composition. 11. A method for diagnosing cancer comprising the immunoreactive compound according to any one of claims 1 to 9. comprising at least one diagnostically useful group attached thereto. , the above composition.